9-(1,3-Dihydroxy-2-propoxymethyl)guanine as antiviral agent

ABSTRACT

The compound 9-(1,3-dihydroxy-2-propoxymethyl)guanine and the pharmaceutically acceptable salts thereof are useful as antiviral agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to 9-(1,3-dihydroxy-2-propoxymethyl)guanine andpharmaceutically acceptable salts thereof which are useful as antiviralagents. The invention also relates to a pharmaceutical compositioncontaining the above compound in combination with a suitable non-toxicexcipient, the composition being useful in combatting viral infections.The invention also relates to a process for preparing the compound ofthe invention.

2. Related Disclosure

Viral infections are widespread and result in a wide variety ofsymptoms. Some viral infections are easily overcome by the body'sdefense mechanism while others are of a more serious nature leading topermanent damage, e.g., blindness, and even to death. One such family ofviruses which may cause serious infections is the Herpes virus group.

The drugs presently used to treat viral infections are ineffective inmany cases or, if effective, are needed in large and/or continuousdosages which produce serious side-effects and/or toxicity. Thereforethere is a need for an effective antiviral agent which is effective atlower dosages than the presently available drugs, thus diminishing thechance of possible side-effects and toxicity.

U.S. Pat. No. 4,199,574 discloses compounds represented by the followinggeneric formula: ##STR1## wherein X is sulphur or oxygen, R¹ ishydrogen, halogen, hydroxy, alkoxy, azide, thio, alkylthio, amino,alkylamino or dialkylamino; R² is hydrogen, halogen, alkylthio,acylamino, amino or azide; R³ is hydrogen, straight or branch chain orcyclic alkyl, hydroxyalkyl, benzyloxyalkyl or phenyl; R⁴ is hydrogen,hydroxy or alkyl; R⁵ is hydrogen, hydroxy, amino, alkyl, hydroxyalkyl,benzyloxy, benzoyloxy, benzoyloxymethyl, sulphamoyloxy, phosphate,carboxypropiamyloxy, straight chain or cyclic acyloxy having from 1 to 8carbon atoms e.g., acetoxy or substituted carbamoyl group of formulaNHCO--Z wherein Z is alkyl, aryl or aralkyl optionally substituted byone or more of sulphonyl, amino, carbamoyl or halogen; R⁶ is hydrogen oralkyl, provided that when X is oxygen and R², R³, R⁴, and R⁶ arehydrogen, R¹ is not amino or methylamino when R⁵ is hydrogen or hydroxy,or a salt thereof.

The class of compounds represented by the above formula and thepharmaceutically acceptable acid addition salts thereof are described toexhibit antiviral activity. See also Tetrahedron Letters, 21, 327-30(1980).

SUMMARY OF THE INVENTION

It has now been discovered that, surprisingly, one specific compound,9-(1,3-dihydroxy-2-propoxymethyl)guanine of the above class and itssalts is a particularly active antiviral agent. The selective activityof this compound is highlighted when the compound is compared with thestructurally most similar compounds disclosed in U.S. Pat. No. 4,199,574in an antiviral assay as shown in detail in Example 5.

The first aspect of the present invention is the compound9-(1,3-dihydroxy-2-propoxymethyl)guanine which may be represented by theformula ##STR2## and the pharmaceutically acceptable salts thereof.

Another aspect of the invention relates to pharmaceutical compositionsfor antiviral use comprising the compound of the instant invention and asuitable carrier.

A further aspect of the invention is a method of treating viralinfections consisting of administering a compound of the presentinvention of a composition containing same.

Yet another aspect of the invention is a process for preparing thecompound of formula (I) which comprises reacting a protected guaninederivative such as N², 9-diacylguanine wherein the acyl group containsone to eight carbon atoms with a compound of the formula ##STR3##wherein R is a protecting group such as benzyl optionally substitutedwith alkoxy, preferably methoxy or alkyl, preferably methyl and R¹ ishydrogen or alkyl of one to four carbon atoms, preferably methyl, andthen removing the protective groups.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENT

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated.

"Pharmaceutically acceptable salts" refers to those salts which possessthe biological effectiveness and properties of the free compound andwhich are not biologically or otherwise undesirable. The salts may beeither the acid addition salts or alkali metal salts. Suitable acids forsalt formation are inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as trifluoroacetic acid, menthanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid and the like. Suitablebases for salt formation are alkali metal bases such as alkali metalhydroxides, e.g., sodium hydroxide, potassium hydroxide and the like.

The compound of the present invention characterized by formula (I)above, and the pharmaceutically acceptable salts thereof, aredistinguished by the discovery that the 1,3-dihydroxy-2-propoxymethylgroup substituted at the 9-position of the guanine nucleus provides,surprisingly, a highly active compound.

UTILITY AND ADMINISTRATION

The subject compound of formula (I) and the pharmaceutically acceptablesalts thereof exhibit potent antiviral activity. For example, thecompound of the present invention exhibits excellent activity againstHerpes Simplex virus I and II and related viruses such ascytomegalovirus, Epstein-Barr virus and varicella Zoster virus.

Pharmaceutical compositions containing the subject compound appropriatefor antiviral use are prepared by methods and contain excipients whichare well known in the art. A generally recognized compendium of suchmethods and ingredients is Remington's Pharmaceutical Sciences by E. W.Martin, (Mark Publ. Co., 15th Ed., 1975).

The compound of the invention may be administered parenterally (forexample, by intraveneous, intraperitoneal or intramuscular injection),orally, or rectally, depending on whether the preparation is used totreat internal or external viral infections.

For internal infections the compositions are administered orally orparenterally at dose levels, calculated as the free base, of about 0.1to 300 mg/kg, preferably 1.0 to 30 mg/kg of mammal body weight, and areused in man in a unit dosage form, administered one to four times dailyin the amount of 1 to 250 mg per unit dose. For oral administration,fine powders or granules may contain diluting, dispersing and/or surfaceactive agents, and may be presented in a draught, in water or in asyrup; in capsules or sachets in the dry state or in a non-aqueoussolution or suspension, wherein suspending agents may be included; intablets, wherein binders and lubricants may be included; or in asuspension in water or a syrup. Where desirable or necessary, flavoring,preserving, suspending, thickening or emulsifying agents may beincluded. Tablets and granules are preferred, and these may be coated.

For parenteral administration or for administration as drops, as for eyeinfections, the compounds may be presented in aqueous solution in aconcentration of from about 0.1 to 10%, more preferably about 0.1 to 7%.The solution may contain antioxidants, buffers, etc.

Alternatively for infections of the eye, or other external tissues,e.g., mouth and skin, the compositions are preferably applied to theinfected part of the body of the patient topically as an ointment,cream, aerosol or powder, preferably an an ointment or cream. Thecompounds may be presented in an ointment, for instance with a watersoluble ointment base, or in a cream, for instance with an oil in watercream base, in a concentration of from about 0.01 to 10%; preferably 0.1to 7%, most preferably about 0.5% w/v. Additionally, viral infections ofthe eye, such as Herpetic keratitis may be treated by use of a sustainedrelease drug delivery system as is described in U.S. Pat. No. 4,217,898.

The exact regimen for administration of the compounds and compositionsdisclosed herein will necessarily be dependent upon the needs of theindividual subject being treated, the type of treatment and, of course,the judgement of the attending practitioner.

PREPARATION

The compound of formula (I) may be prepared by the following reactionscheme. ##STR4##

The compound of formula (III) is prepared by adding epichlorohydrin (II)dropwise to a solution of an alkali metal salt, preferably the sodiumsalt, of optionally substituted benzyl alcohol in a solvent such asdimethylformamide, dimethylacetamide, hexamethylphosphoramide,dimethylsulfoxide, sulfolane, tetrahydrofuran, and dioxane at atemperature of about 0° C. to 100° C., preferably at about 15° C. to 40°C. The reaction mixture is stirred from about 10 hours to 24 hours,preferably from about 12 hours to 18 hours at a temperature of about 0°C. to 100° C., preferably from about 20° C. to 50° C.

Compound of formula (III) is chloromethylated to compound of formula(IV) by bubbling dry hydrogen chloride gas in a solution of the compoundand paraformaldehyde dissolved in a halogenated hydrocarbon solvent suchas dichloroethane, chloroform, dichloromethane, and1,1,2-trichloroethane cooled to a temperature of about 0° C. to 25° C.,preferably at a temperature of about 0° C. The hydrogen chloride gas isadded over 30 minutes to 3 hours, preferably over 1 hour to 2 hoursuntil the paraformaldehyde dissolves. The solution is held at atemperature from about 0° C. to 10° C. for about 12 hours to 48 hours,preferably from about 0° to 5° for about 16 hours to 24 hours.

Compound of formula (V) is prepared by reacting an alkali metal acetatesuch as sodium acetate with compound of formula (IV) dissolved in asolvent such as dimethylformamide, tetrahydrofuran, dimethylacetamide,hexamethylphosphoramide, dimethylsulfoxide, sulfolane, and dioxane at atemperature of about 0° C. to 45° C., preferably from about 0° C. to 25°C. The solution is stirred from about 5 to about 24 hours, preferablyfrom about 10 hours to about 18 hours at a temperature of about 10° C.to about 30° C., preferably at a temperature of about 15° C. to 25° C.

Compound of formula (VII) is prepared by heating guanine (VI) withacetic anhydride, neat, at reflux for about 10 to 24 hours, preferablyfor about 12 to 18 hours.

N²,9-Diacetylguanine of formula (VII) is reacted with compound offormula (V) to form compound of formula (VIII) neat or in a solvent suchas dioxane, sulfolane and the like in the presence of a catalytic amountof an acid such as bis(p-nitrophenyl)phosphate, toluene sulfonic acid,methylphosphonic acid or dichloroacetic acid, preferablybis(p-nitrophenyl)phosphate at a temperature of about 75° C. to 200° C.,preferably at about 110° C. to 180° C. The reaction is generally carriedout using 0.8 moles to 1.2 moles of compound of formula (V) to one moleof compound of formula (VII).

The benzyl protecting groups are removed from compound of formula (VIII)by catalytic hydrogentation to form compound of formula (IX). A catalystsuch as palladium on carbon in a slurry is added to a solution ofcompound of formula (VIII) dissolved in a solvent such as aqueousmethanol. Hydrogen is added to the solution at a pressure of 15 psi to200 psi, preferably at a pressure of 30 psi to 80 psi.

Compound of formula (I) is prepared by deacetylating compound of formula(IX) with a base such as ammonia dissolved in an alcohol such asmethanol. A solution of compound of formula (IX) and the base is stirredfor about 5 hours to 36 hours, preferably for about 10 hours to 24 hoursat a temperature of about 10° C. to 30° C., preferably at a temperatureof about 15° C. to 25° C.

The following specific description is given to enable those skilled inthe art to more clearly understand and practice the invention. It shouldnot be considered as a limitation upon the scope of the invention butmerely as being illustrative and repesentative thereof.

PREPARATION I Preparation of 1,3-Di-O-benzylglycerol

Sodium hydride (100 g (50% dispersion in mineral oil), 2.08 mol) waswashed twice with 1 l of hexane then dried under nitrogen. Drydimethylformamide (1.5 l) was added. Benzyl alcohol (400 ml) was thenadded at such a rate to keep the temperature below 50° C. The additiontook 2 hours. Epichlorohydrin (92.5 g, 1 mol) was then added dropwiseover 0.5 hour with ice cooling in order to keep the temperature below40° C. The solution was next stirred for 16 hours at 21° C. then for 2.5hours at 50° C. The dimethylformamide was then removed by evaporation atreduced pressure. The oily residue was dissolved in 2.5 l diethyl ether.The organic solution was washed with 2 l of water, 2 l of 2%hydrochloric acid, 2 l of 1% sodium bicarbonate, and 1 l of brine, driedover sodium sulfate, and concentrated to a brown oil. Distillation gave147.8 g of 1,3-di-O-benzylglycerol (bp 170°-180° C./1 torr).

PREPARATION II Preparation of 1,3-Di-O-benzyl-2-O-chloromethylglycerol

Dry hydrogen chloride gas was bubbled for 1.5 hours into a solution of1,3-di-O-benzylglycerol from Preparation I (15 g, 55 mmol) andparaformaldehyde (3.3 g, 110 mmol) in 175 ml of 1,2-dichloroethane at 0°C. The solution was then stored in a stoppered flask for 21 hours at 4°C. Next, the solution was dried over magnesium sulfate with warming to21° C. then filtered and concentrated to give 17.5 g of1,3-di-O-benzyl-2-O-chloromethylglycerol.

PREPARATION III Preparation of 2-O-Acetoxymethyl-1,3-di-O-benzylglycerol

To a solution of 1,3-di-O-benzyl-2-O-chloromethylglycerol fromPreparation II (17.5 g, 55 mmol) in 400 ml of dimethylformamide at 0° C.under a drying tube was added sodium acetate (6 g). The solution wasthen warmed to 21° C. and magnetically stirred for 15 hours. The solventwas removed by evaporation at reduced pressure and the oily residuedissolved in 1 pound of diethylether. The ether solution was washed oncewith 750 ml of water, two times with 250 ml of water, and once with 250ml of brine, dried over sodium sulfate and concentrated to give 19 g of2-O-acetoxymethyl-1,3-di-O-benzylglycerol as an oil.

PREPARATION IV Preparation of N²,9-Diacetylguanine

Guanine (20 g, 0.132 mol) was combined with 300 ml of acetic anhydrideand the mixture heated at reflux for 16 hours. The mixture was cooledand the excess acetic anhydride removed by evaporation at reducedpressure. The residue was recrystallized from dimethyl sulfoxide to give25.6 g of N²,9-diacetylguanine.

EXAMPLE 1 (A) Preparation of N²-Acetyl-9-(1,3-dibenzyloxy-2-propoxymethyl)guanine

N²,9-Diacetylguanine from Preparation IV (15.61 g, 66 mmol),2-O-acetoxymethyl-1,3-di-O-benzylglycerol from Preparation III (19 g, 55mmol), and bis(p-nitrophenyl)phosphate (0.5 g) were stirred togetherwith 150 ml of diethylether. The solvent was removed by evaporation andthe residue heated in a 175° C. oil bath for 1.5 hours under a stream ofnitrogen. Column chromatography eluting with 1:9 methanol/methylenechloride followed by recrystallization from ethyl acetate afforded 4.76g of N² -acetyl-9-(1,3-dibenzyloxy-2-propoxymethyl)guanine, mp 145°-146°C.

(B) Preparation of N² -Acetyl-9-(1,3-dihydroxy-2-propoxymethyl)guanine

To a solution of N² -acetyl-9-(1,3-dibenzyloxy-2-propoxymethyl)guanine(4.62 g, 9.67 mmol) in 150 ml of methanol plus 40 ml of water was added20% palladium hydroxide on carbon as a slurry in 10 ml of water. Themixture was hydrogenated on a Parr hydrogenator at 60 psi of hydrogenfor 38 hours then filtered through celite and concentrated to a whitesolid. Recrystallization from methanol/ethyl acetate gave 1.4 g of N²-acetyl-9-(1,3-dihydroxy-2-propoxymethyl)guanine, mp 205°-208° C.

The mother liquor was further reduced with 10% palladium on carbon (1 g)in 150 ml of methanol plus 50 ml of water at 60 psi for 47 hours. Thetotal yield of N² -acetyl-9-(1,3-dihydroxy-2-propoxymethyl)guanine was2.11 g.

(C) Preparation of 9-(1,3-Dihydroxy-2-propoxymethyl)guanine

N² -Acetyl-9-(1,3-dihydroxy-2-propoxymethyl)guanine (721.9 mg, 2.4 mmol)was stirred with 50 ml of methanolic ammonia solution (methanolsaturated with ammonia at 0° C.) for 17 hours at 21° C. The solution wasconcentrated to a white solid and the residue recrystallized frommethanol to give 582.3 mg of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine,mp 250° C. d.

EXAMPLE 2

9-(1,3-Dihydroxy-2-propoxymethyl)guanine was dissolved in a solution ofwater containing one mole equivalent of sodium hydroxide. The solutionwas then lyophilized to give 9-(1,3-dihydroxy-2-propoxymethyl)guaninesodium salt as a white powder.

EXAMPLE 3

The sodium salt of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine wasdissolved in a minimum amount of water and dilute hydrochloric acid wasadded to adjust the pH to 7. 9-(1,3-Dihydroxy-2-propoxymethyl)guanine,m.p. 250° C. d crystallized from the solution.

EXAMPLE 4

The following example illustrates the preparation of representativepharmaceutical formulations containing an active compound of Formula(I).

    ______________________________________                                        A. Topical Formulation                                                        ______________________________________                                        Active compound          0.2-2  g                                             Span 60                  2      g                                             Tween 60                 2      g                                             Mineral oil              5      g                                             Petrolatum               10     g                                             Methyl paraben           0.15   g                                             Propyl paraben           0.05   g                                             BHA (butylated hydroxy anisole)                                                                        0.01   g                                             Water  qs                100    ml                                            ______________________________________                                    

All of the above ingredients, except water, are combined and heated at60° C. with stirring. A sufficient quantity of water at 60° C. is thenadded with vigorous stirring to provide 100 g of the cream formulationwhich is then cooled to room temperature.

The following formulation is useful for intraperitoneal andintramuscular injection.

    ______________________________________                                        B. IP and IM Formulation                                                      ______________________________________                                        Active compound         0.5    g                                              Propylene glycol        20     g                                              Polyethylene glycol     20     g                                              Tween 80                1      g                                              0.9% Saline solution  qs                                                                              100    ml                                             ______________________________________                                    

The active compound is dissolved in propylene glycol, polyethyleneglycol 400 and Tween 80. A sufficient quantity of 0.9% saline solutionis then added with stirring to provide 100 ml of the I.P or I.M solutionwhich is filtered through a 0.2 micron membrane filter and packagedunder sterile conditions.

The following formulation is useful for intravenous injection.

    ______________________________________                                        C. I.V. Formulation                                                           ______________________________________                                        Active compound        0.5    g                                               0.9% Saline solution   100    g                                               ______________________________________                                    

The active compound is added to 100 ml of 0.9% saline solution withstirring to provide 100 ml of I.V. solution which is filtered through a0.2 micron membrane filter and packaged under sterile conditions.

    ______________________________________                                        D. Tablet Formulation                                                                          Parts by weight                                              ______________________________________                                        Active compound    200                                                        Magnesium stearate 3                                                          Starch             30                                                         Lactose            116                                                        PVP (polyvinylpyrrolidone)                                                                       3                                                          ______________________________________                                    

The above ingredients are combined and granulated using methanol as thesolvent. The formulation is then dried and formed into tablets(containing 200 mg of active compound) with an appropriate tablettingmachine.

EXAMPLE 5

The exceptional antiviral activity of the compound of the invention isillustrated by the following assay procedures:

The Herpes simplex virus 2 strain G for infection was prepared in HEp-2cell cultures. Virus was adsorbed for 1 hour, fresh media was placed onthe cells, and they were incubated at 35° C. until all cells wereinfected. The cell suspension was frozen at -70° C., thawed, andcentrifuged to remove cell debris. The supernatant fluid was aliquotedand stored frozen at -70° C. until use. A 10⁶.7 dilution of thesupernatant fluid produced a 50% cell culture infective dose (CCID₅₀) inHEp-2 cells and a 10³.7 dilution produced a 50% lethal challenge (LC₅₀)in mice.

Groups of 20 Swiss Webster female mice (15-17 gm), were challenged byintraperitoneal route using 0.2 ml of EMEM containing 10 LC₅₀ /mouse ofvirus. Mice challenged with 10⁰.5 more or less virus than the 10 LD₅₀challenge served as a virulence control to assure the model was workingproperly.

Treatment with test compounds began 6 hours post-challenge. The mice,divided into groups of 20, were administered the compounds in salines.c. at 5 mg/kg, 10 mg/kg and 20 mg/kg. One group of 20 mice was used asa control group and administered saline s.c. The treatment was repeatedat 24, 48, 72 and 96 hours post-challenge.

Test compounds were the compound of the invention, and three compoundsdisclosed in U.S. Pat. No. 4,199,574 having structural similarity toportions of the compound of the invention.

    ______________________________________                                                            Survivers (out of 20)                                                         Days Post Challenge                                       Test Compound   Dosage    12      14   21                                     ______________________________________                                        Untreated Controls            0     0    0                                    9-(1,3-dibenzoxy-2-propoxy                                                                    5     mg/kg   2     2    2                                    methyl)adenine  10    mg/kg   1     0    0                                                    20    mg/kg   1     1    1                                    9-(1,3-dibenzoxy-2-propoxy                                                                    5     mg/kg   2     0    0                                    methyl)-6-mercaptopurine                                                                      10    mg/kg   0     0    0                                                    20    mg/kg   1     0    0                                    9-(1-hydroxy-2-ethoxy                                                                         5     mg/kg   1     1    1                                    methyl)guanine  10    mg/kg   3     3    3                                                    20    mg/kg   5     4    4                                    9-(1,3-dihydroxy-2-propoxy                                                                    5     mg/kg   15    14   14                                   methyl)guanine  10    mg/kg   19    18   18                                                   20    mg/kg   19    18   15                                   ______________________________________                                    

In the above results, a comparison between the number of survivinganimals administered the compound of the invention and the number ofsurviving animals administered the three prior art compounds clearlydemonstrates the markedly superior antiviral activity of our compound.

What is claimed:
 1. The compound9-(1,3-dihydroxy-2-propoxymethyl)guanine and the pharmaceuticallyacceptable salts thereof.
 2. The compound9-(1,3-dihydroxy-2-propoxymethyl)guanine.
 3. The sodium salt of9-(1,3-dihydroxy-2-propoxymethyl)guanine.
 4. The antiviralpharmaceutical composition which comprises a pharmaceutically acceptablecarrier and an effective amount of9-(1,3-dihydroxy-2-propoxymethyl)guanine or a pharmaceuticallyacceptable salt thereof.
 5. Method of treating viral infections in amammal having a viral infection which comprises administering aneffective amount of 9-(1,3-dihydroxy-2-propoxymethyl)guanine or apharmaceutically acceptable salt thereof or a composition containingsame as an active ingredient.